Category Archives: RNA

Most of the time, biologist in the lab aren’t working with things that they can see. You need to look at cell under a microscope, proteins using X-Ray crystallography (for example), and DNA and RNA by various methods like gel electrophoresis or cell staining (see more about visualizing DNA here). Many experiments require scientists to manipulate DNA, RNA or proteins in small liquid volumes to do experiments to understand the sequence of these molecules, to discover what other molecules they bind to, or to change them to figure out how they work in cells. To manipulate these small volumes, we use a tool called a pipette (shown at left) – also called a micropipette to be even more precise. It’s similar in concept to the baster you have in your kitchen that sucks up liquid when basting your turkey, except it’s for much smaller volumes and is exquisitely precise.

Pipettes come in different sizes, and the size of the pipette determines the volume that they can dispense (see picture at right). They dispense microliter (abbreviated as “ul”) volumes of liquid. To give you an idea of scale, 1 drop of water is about 50ul. One milliliter (ml) is1000ul, which is the largest micropipette volume you can dispense, and there are about 44ml in a shot of vodka. These are small volumes we are talking about!!

The sizes shown to the right dispense up to 2ul (called a “p2”), 200ul (“p200”), or 1000ul (“p1000”). By spinning the plunger on the pipette, you can change the volume that is dispensed within the range that the pipette allows. You then put disposable tip to the end of the pipette and suck up that volume of liquid. As an example, check out the photo below of the p1000 dispensing 600ul of water and the p200 dispensing 20ul of water. The image to the left is the pipette tip filled with that volume and to the right is the water in a tube. This type of tube is used every day in the lab and is called an eppendorf tube. IT can hold 1.5ml (or 1500ul) of water. These small plastic tubes have attached caps that can snap the tube closed to allow you to mix the contents or do whatever is needed for the experiment (like heat it up, cool it down, spin the contents, etc). And if you’re wondering why they are called “eppendorf” tubes, it’s after the name of a German company called Eppendorf that is one manufacturer of this kind of tubes (similar to how the brand Kleenex is now synonymous with tissues)

Maybe you’re wondering why scientists have to use such small volumes? One reason is because there isn’t a large amount of DNA or RNA or proteins in cells, so when it’s isolated, it doesn’t take up a large volume. As an example, if you isolate the genomic DNA from 5 million cells, you would isolate 10-30ug of DNA. That’s MICRO grams of DNA – or 0.00003 grams. As comparison, a grain of salt weighs more than 1200x more than the amount of DNA that you would isolate from 5 million cells! And this is all in just 50ul of liquid. Very small amounts! The good news is that working in large volumes isn’t really needed because most experiments don’t need a large quantity of materials to get the answer. Not to mention that working with larger volumes for experiments would be more expensive. If you are doing an enzymatic reaction, a larger volume reaction would require more enzyme. To get an idea of what this would cost, a very common reaction cuts DNA with restriction enzymes, which are essentially super-specific DNA scissors. These restriction enzymes cost several hundred dollars per tube and you use 0.5-1ul for every 50ul reaction. If you did a reaction in a large test tube of 10ml, you’d need 200 times more enzyme and would go through hundreds or thousands of dollars if enzyme for each experiment. Labs are not that rich!

Fortunately scientists have developed techniques and equipment, like the micropipette, that can manipulate, detect and analyze incredibly small amount of DNA or RNA or proteins. For most bench scientists, learning how to use a micropipette is done on day one (I learned how to use one in high school at City Lab) and pretty soon becomes second nature. In grad school, I pipetted so much that there were days I’d go home with a sore thumb or pipetting calluses. However all this practice did pay off. I picked up a pipette for the first time in 8 years a few weeks ago to start doing experiments in my new lab. After all these years, it was just like riding a bike – or in this case just like “using a pipette”.

I was at a party last night telling my non-scientist friends about my week at work. For the first time since I left my post-doc 8 years ago, I spent multiple days in the lab doing experiments!! We had a few drinks at this point, and in their lapse of judgement (thinking I may not give them a descriptive answer at a party) they asked me what my experiments were about. I did not disappoint, and told them all about my experiments.

This party was a BBQ and even though I’m a vegetarian, most of my friends are huge fans of meat. So I started by asking them about steak. If you bought a steak that you weren’t going to throw on the grill right away, you might freeze it and cook it later. When you take it out of the freezer, how do you know if the steak is still of high quality? Maybe it sat on the counter overnight before being put in the freezer. Maybe the freezer lost power during a storm, the steak defrosted and when the power came back on, the steak froze again. Maybe the steak wasn’t even good when it got to your house – maybe it sat on the butcher’s counter for a few days, forgotten about, before being put into the display case for you to buy. It may be nearly impossible to tell by just looking at your frozen steak if any of these events happened and whether or not these events would affect the steak’s quality.

This is something that we deal with daily as biobankers. To remind you, we collect human tumor samples for researchers to use to better understand diseases and to develop improved treatments. Ideally these tissue samples are very quickly frozen (as described in this blog post) and kept frozen. Like the steak, there are a lot of “variables” that may affect the quality of the tumor tissue sample – including the same issues that a steak may have – like sitting at room temperature for too long or freezing and then thawing and then freezing again. And also similar to the steak, it’s nearly impossible to tell by just looking at the tissue sample if any of these things have happened. Except, instead of affecting whether or not your dinner tastes good, with tumor tissue samples, research results that are essential for drug or biomarker development for brain tumors are affected.

How do we handle this little problem? We have to use a proxy for quality – something that we can analyze directly that will tell us whether or not the tissue sample is of good enough quality to be used for certain research purposes. In this case, the proxy is RNA. RNA is a great molecule to look at because it’s found in every cell, but it’s also unstable because of its natural enemy RNases. When these RNases are active (when the tissue sample is warm or at room temperature) they will function like little PacMans to chomp on the RNA, turning it into smaller and smaller pieces. When frozen, the RNases are inactive and can’t chomp on RNA. So if you look at how chomped up/small the RNA is in tissue samples, you can figure out whether the quality of the sample is good for your experiments.

A tiny filter (seen as the white line inside the pink tube) binds to the RNA.

This is what I did last week in the lab. How did I do this? First, I had to get the RNA out of the tissue sample. To do this, you have to separate the cells from one another, which essentially like grinding up a steak in a meat grinder. You then have to bust open the cells to get to the RNA. You do that by adding something that works a lot like soap that opens up the outer coating of the cells. From there, you can isolate the RNA by adding ethanol (an alcohol) that makes the RNA no longer dissolved in the liquid (what we call “precipitating” the RNA). From there we isolate the RNA on a column – exactly like the pink one you see on the right. The white line inside the pink tube is like an RNA filter that traps RNA while letting all the other cell bits flow through. Then you use water to get the RNA out of the filter. How much RNA do you get at the end? Imagine an eye drop worth of liquid that contains 10 micrograms of RNA. That’s 0.0001 grams and for comparison, a grain of rice weighs 0.015 grams. It’s not a lot, but it’s enough to know if your sample is good or not.

These ScreenTapes can analyze 16 RNA samples at one time. They are about the size of a long box of a glass slide or a ling box of matches

You still can’t “see” the RNA to know if it is all in one piece or if it’s been chomped up by RNases just by looking at this tiny bit of liquid in a tube. You still have to analyze it, and to do this, I used a cute little machine called a TapeStation (I honestly have no idea why it’s called this, since the “ScreenTapes” that you use for the analysis – in the photo at left – look nothing like Tapes to me). This machine separates the RNA based on size (you can see that by the black lines in the image below). There are two main sizes it looks at – these separate into to peaks (called 16S and 28S). If these RNAs have been chomped up, there won’t just be two peaks (or two black lines), but lots of little peaks of smaller sizes. This will indicate that the RNases were activated for some reason and the quality isn’t as good. In my experiments, the results were awesome and the RNA was of good enough quality for most experiments. It also meant that the tumor tissue was handled really well when it was collected and didn’t sit on the counter or get thawed and frozen again, for example.

The two peaks are a graphical image of the separation of the RNA shown by the dark lines on the right.

Now you may be wondering, “what about my steak?” I honestly cannot encourage you to do this level of analysis to see if your steak’s RNA is high quality. Then again, you just want to eat the steak – not do thousands of dollars of important experiments with it. So let’s just consider the steak a useful scientific analogy and go start your grill – I’ve heard there are some steaks in your freezer.

Thanks to Kelli and Alia for asking me what experiments I’ve been doing and inspiring this blog post.

If you’re into yoga, you may be very familiar with “OM” or if you’re an electrician with “ohms”, but in science, we use “-ome” in a very different way. To start off, let’s give a silly example: the studentome (which I’m fairly sure does not actually exist). This would be the study of all “students” in a certain place. Maybe we’re interested in all of the students in a particular high school or college, and they can be categorized and better understood by looking at the distribution of their ages, their heights, their grade level, their clothing style, etc. The studentome would be different in an inner city school compared to a private Catholic school, and understanding these differences could help to improve or change aspects of the studentome in a certain place. The study of the studentome, would be called studentomics.

So what does this “-ome” mean? In Greek: “-ome” means “all” or “complete”. So whenever scientists put “-ome” at the end of a word, they are talking about all of something (like with the “studentome” all of the students), and in biology this usually is referring to all of something in an organism or a particular cell type. It’s not clear when scientists started using words ending in “-ome”, though the word biome was coined in the early 1900s. The modern usage likely started in the early 1990s as technologies, like computers and DNA sequencing, allowed scientists to study all of something in an organism with more ease. A derivative of the “-ome” is “-omics”, which is the study of all of something in an organism or a cell. This terminology is so common, there is even a wiki dedicated to “-omes” and “-omics” here.

Let’s explore some of the common “-omes” (you can find a more comprehensive list of scientific “-omes” here).

Genome: The most famous “ome” is the genome. The genome refers to all of the DNA in an organism. In humans, this includes all 23 pairs of chromosomes (number 1-22 and the two sex chromosome, XX if you are female and XY if you are male). Scientists study the genome to understand the genetic blueprint of DNA because DNA codes for proteins, which are the functional machines that do everything in a cell.

Transcriptome: For DNA to make a protein, the DNA needs to be “transcribed” in RNA first (read more details about this process here). All of the RNA in a cell is called a transcriptome. Scientists study the transcriptome because not all DNA is “turned on” to make proteins in every cell. This helps explain why certain cells look different (skin cells look different than eye cells) and have a different function (skin cells provide a barrier from the environment and specialized eye cells allow you to see).

Proteome: And this brings us to the proteome, which is all of the proteins in a cell or organism. Since proteins are what’s actually doing stuff in a cells, by understanding what proteins are present in certain cells, scientists are able to better understand how those cells function. And in the case when there are problems, for example in cancer cells, it can help understand why there is a problem and possible ways to fix it.

Interactome: Proteins interact with one another in a variety of ways. The interactome maps all of these interactions. The interactome is also different in different cell types because the proteins expressed are different in different cell types, so there are many interactomes

Metabolome: Even though proteins are the machinery in a cell, there are lots of other small molecules and chemicals called metabolites. For example, glucose is a metabolite that is broken down to produce energy. All of the metabolites in an organism are called the Metabolome.

And there are hundreds more of these “-omes”! This “omeome” (originally and jokingly coined here) has even seeped into more popular culture. For example, the Facebookome, described as “The totality of facebook social network connections and nodes information such as people’s names, relationships, and multimedia contents.” Although it may seem to be an unnecessary wordy trend, these “-omes” and “-omics” are necessary for scientists to better understand health and disease.

You JUST HEARD about the central dogma last week, but it’s so important, that I think it’s worth a reminder – and the best kind of reminder is one in a video.

I also think it’s important to point out that there are a lot of details involved in the processes of transcription and translation. And I don’t say this to make you feel badly that you don’t know the details, but rather to point out that these different steps all provide the cell with the opportunity to fine tune to protein production machine. Back to our steering wheel analogy – you can take away the blueprint for part of the day so that the person can’t make steering wheels. In this case, it would mean that something is making the DNA unavailable for transcription. Or you could tell the person making the steering wheels to go on break. This would be like getting rid of the mRNA. Or you could take away some of the pieces needed to make the steering wheel, which would be like decreasing the production of an amino acid. All of these scenarios happen!

I just want you to start thinking about how these steps can be “regulated” because then when we start talking about “deregulation” (meaning, when things aren’t regulated properly), you’ll understand that there can be many ways for this to happen.

Genes are pieces of DNA that code for a protein. That sounds great…but how does it do this? DNA is made out of nucleotides (A, T, C, and G) and proteins are made out of amino acids. To “crack the code” and transform the DNA code into the protein code, you need an intermediary. That’s a molecule called RNA! messenger RNA (or mRNA, for short) to be precise, because it is the message that communicates the information from the DNA to the protein.

Let’s revisit our car blueprint analogy to discuss the role of mRNA. If DNA is the blueprint for the steering wheel, and the protein is the steering wheel, mRNA is the person in the middle who reads the blueprint and builds the steering wheel.

So let’s see how this actually works. The first step is to understand what RNA is actually made out of. DNA stands for DeoxyriboNucleic Acid and RNA stands for RiboNucleic Acid, and if they sound like they are related, you are correct. Here are the three main differences

DNA is double-stranded and RNA is single stranded (see the photo here)

DNA is made from A, T, C, and G and RNA uses A, U (standing for uracil), C, and G

In DNA, A always matches with T, whereas with RNA, A always matched with U

Because they are made up of similar components, the cell can copy the code from the DNA into the single-stranded mRNA molecule. Also because these two molecules are related, this process is call transcription – like what you would do when copying the words in a book from one place (DNA) to another (mRNA).

The mRNA is then what codes for the protein. It does this just like you would expect any code to – using a key. In this case, each set of 3 nucleotides codes for an amino acid. The cells have machinery (called a ribosome) that “reads” the mRNA strand. The combination of “ATG” always tells the ribosome to START making a protein. It then reads the three digit code, adding the appropriate amino acids along the way, until it reads one of the three codons that tells the ribosome to STOP making protein. At this point, the protein can fold up to make the 3D structure so it will function. Because this process takes nucleotides and codes them into amino acids (a totally different molecule), this process is called translation. Just like translating a book from English into French, this process translates mRNA into a protein.

Wow what a lot to take in! This process from DNA (the gene) to mRNA to protein is called the “central dogma” of molecular biology. This process was originally described in 1956 by Francis Crick (the same guy who along with Watson discovered the structure of DNA). Why is it called this? Probably because when it was described, they realized that this was a fundamental process for all life. It’s also important for us to know for so many reasons. If you know that DNA codes for RNA which codes for protein, any changes in this process can result in a protein that isn’t quite right. What if you have change the code of the DNA? This will change the protein! What if you change the amount of the mRNA? You will change the amount of protein! And what can this do? Cause problems in your cell that cause disease!